The last two decades have seen an increase in the availability of drugs for the treatment of a variety of diseases, the identification of a wide variety of compounds associated with pathogens, and the need to detect small amounts of contaminants in the environment and industrial effluents. Thus, assays are needed and used for detecting the presence of analytes in test samples in fields such as clinical and forensic medicine, environmental testing, food quality assurance, and drug use testing and related areas.
A variety of diagnostic tests have been developed for the routine identification or monitoring of physiological and pathological conditions (e.g., pregnancy, cancer, endocrine disorders, infectious diseases) using a variety of biological samples (e.g., urine, serum, plasma, blood, saliva) and environmental samples (e.g., natural fluids and plant effluents). Many of these diagnostic tests are based on the highly specific interactions between specific binding pairs. Examples of such binding pairs include antigen/antibody, hapten/antibody, lectin/carbohydrate, apoprotein/cofactor and biotin/streptavidin. Furthermore, many of these tests involve devices (e.g., lateral-flow test strips, flow-through tests) with one or more of the members of a binding pair attached to a mobile or immobile solid phase material such as latex beads, glass fibers, glass beads, cellulose strips or nitrocellulose membranes. The attachment of molecules such as antibodies, antigens, biotin or streptavidin to a solid phase material normally involves either passive adsorption or covalent bonding.
Current methodology for the attachment of molecules (e.g., peptides and proteins) to microporous solid phase material (e.g., nitrocellulose in lateral-flow and flow-through devices) often involves the passive adsorption of molecules to the solid phase material. The interaction between the attached molecules and solid phase material is primarily hydrophobic in nature, based on van der Waals forces, or due to hydrogen bonding. Passive adsorption is subject to several limitations. To consistently maintain their protein-interactive properties, solid phase materials like nitrocellulose membranes must remain hydrated and be stored in a stable environment of controlled humidity and temperature. Furthermore, passive adsorption is not effective in immobilizing small molecules (.ltoreq.1000 Daltons) such as drugs, hormones and small peptides (see Lauritzen et. al., J. Immunol. Methods, 131(2):257-267 (1990)) or nucleic acid polymers in conformations that are conducive to nucleic acid hybridization and palindromic sequence folding. Due to these passive adsorption limitations, small molecules (&lt;1000 Daltons), nucleic acid polymers and some proteins preferably are covalently bound to a solid phase material to increase stability and prevent palindromic sequence folding. It is also preferable to bond the molecule to a microporous solid phase material that has flow properties suitable for lateral-flow applications.
U.S. Pat. No. 3,857,931 shows that peptides and proteins can be chemically bonded to latex particles that have surface carboxyl groups. Carbodiimide can be reacted directly with the carboxyl groups on the latex to form a transient activated intermediate acyl-isourea, which in turn reacts with amino groups on a molecule to form a stable amide bond. This amide bond couples the peptide or protein to the latex particles' surface. A disadvantage of this process is the inability to control the undesirable and indiscriminate reaction of carbodiimide with the carbonyl groups that are also present on proteinic molecules. Thus, carbodiimide-activated carboxyls on a protein may react with amino groups on the protein to cause intra- or inter-protein crosslinking. Crosslinking occurs especially when the protein or peptide contains relatively large amounts of aspartic and glutamic acid, since those amino acids contain free carboxyl groups. Crosslinking can result in conformational or structural distortion of the proteinic molecule that in turn can affect assay sensitivity.
U.S. Pat. No. 4,045,384 states that carboxylated latex can be reacted with a water-soluble carbodiimide and water-solubilized N-hydroxy compound, such as N-hydroxybenzotriazole, to form an active ester latex. This activated ester latex can be reacted with the amino groups of a protein to covalently bond the protein to latex particles via amide bonds in a three-step process that eliminates protein crosslinking side reactions. This process, however, is not suitable for lateral-flow applications.
Amine-bearing solid phase materials can be chemically derivatized with extended-length, heterobifunctional crosslinking reagents to form an activated solid phase material that will form a covalent bond with thiol-bearing peptides or proteins. See Bieniarz et al., U.S. Pat. Nos. 5,002,883 and 5,063,109. Such solid phase materials include those polymers, glasses, and natural products that contain primary, secondary or tertiary amine groups. Also, solid phase materials containing nitrile groups may be reduced to yield amine groups to produce amine-bearing solid phase materials. Amine-bearing solid phase materials, however, are not suitable for lateral-flow applications.
Activated microporous membranes are commercially available for the covalent bonding of molecules that contain amino groups. For example, the UltraBind.TM. membrane (Pall Gelnan Laboratory, Ann Arbor, Mich.) is a polysulfone/polyacrolein-type membrane that has a high concentration of aldehyde active sites available for covalent bond formation with molecules that contain amino groups. See Pemawansa et al., BioTechniques 9(3):352-355 (1990) and Pemawansa et al., U.S. Pat. Nos. 4,824,870, 4,961,852, 5,160,626. Also activated nylon (Biodyne.TM., and Immunodyne.TM., Pall Corporation, Glen Cove, N.Y.) and activated polyvinylidene difluoride (Irnmobilon.TM., Millipore, Bedford, Mass.) also are available for the covalent attachment of proteins as discussed in Gsell et al., U.S. Pat. No . 4,886,836; Marlow et al., J. Immuno, Methods 30 101:133-139 (1987); and Canas et al., Analytical Biochemistry 211:179-182 (1993). Although the membranes identified above are well suited for blotting applications, those membranes have poor flow characteristics and minimal utility for lateral flow applications. Nitrocellulose membranes are generally better suited to lateral flow applications, and many commercially available pore sizes are available. However, in an untreated state, fluid-permeable, microporous solid phase materials, such as nitrocellulose or glass fibers, lack the organic functionality necessary to effect covalent bonding to molecules containing amino groups.
Methods for the covalent bonding of peptides and proteins to activated nitrocellulose have been reported in the scientific literature. For example, a method to covalently bond peptides and proteins through a diaminoalkane spacer to nitrocellulose was developed for immunochemical applications. See Masson et al., Electrophoresis 14(9):860-865 (1993). Also, divinylsulfone, a spacer of ethylenediamine, and glutaraldehyde have been used to produce an activated nitrocellulose. Peptides were attached to this activated nitrocellulose by reaction of the amino group with the free aldehyde groups, forming unreduced Schiff-base bonds. See Lauritzen et al. in the J. Immunol, Methods 131(2):257-267 (1990) and Electrophoresis 14(9):852-859 (1993).